Hypertension or high blood pressure is considered to be one of the main risk factors for Cardio Vascular Diseases (CVD). One of the mechanisms which regulates blood pressure is the renin-angiotensin system. This is a cascade of reactions leading to the formation of angiotensin II, which has a strong vasoconstrictive and hence blood pressure increasing effect. Inhibition of one of the key enzymes in this cascade: Angiotensin I Converting Enzyme (ACE) reduces formation of angiotensin II and thus has a blood pressure lowering effect. Long term human intervention studies have shown regular intake of low amounts of ACE inhibitors reduces CVD by 25% (Gerstein et al. (2000), The Lancet 355, 253-259).
In a placebo-controlled study, the blood pressure lowering effect of VPP and IPP in sour milk was shown in hypertensive humans (Hata, Y et al. (1996), American Journal of Clinical Nutrition 64, 767-771).
A commercially available fermented milk product, which claims to be “suitable for those with mild hypertension” is Calpis sour milk, fermented with Lactobacillus helveticus and Saccharomyces cervisiae, produced by Calpis Food Industry, Japan.
Another commercially available fermented milk product is Evolus produced by Valio, Finland, which claims to be ‘the first European functional food to help lower blood pressure’.
Both fermented milk products are fermented with Lactobacillus helveticus (Lb. helveticus) strains. The products contain bioactive peptides (VPP and IPP) responsible for in vitro ACE inhibition, which are produced by proteolysis of caseins. Compared to other lactic acid bacteria Lb. helveticus is one of the most efficient proteolytic Lactobacillus species.
The breakdown of caseins by the proteolytic system of lactic acid bacteria can be divided into three stages. Initially breakdown of casein is performed by extracellular proteinases, followed by the uptake of di/tri peptides and oligopeptides (4 to 12 amino acidic residues) using specific uptake mechanisms. In the last stage, peptides are further degraded by intracellular peptidases, yielding small peptides and amino acids for bacterial growth. This complicated proteolytic system of lactic acid bacteria is described in Kunji et al., (1996), Molecular Microbiology 27, 1107-1118. A review on the intracellular peptidase system can be found in Christensen et al., (1999), Molecular Microbiology 76, 217-246.
According to EP-A-1016709, it is desired to produce fermented milk with higher content of the lactotripeptides compared to the content of the lactic acid generated in the fermented milk. It provides a Lactobacillus helveticus strain CM4 that in fermentation gives 30-50 μg of tripeptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) per 0.01 g of DL-lactic acid. In table 2 of EP-A-1016709 it is shown that this strain produced 38.5 μg/ml whey of VPP and 23.5 μg/ml whey IPP, which corresponds to an IPPeq value, as defined hereinafter, of 140.
WO 01/034828 discloses a process for producing tripeptides VPP and/or IPP starting from casein using enzymes. In the process casein is treated with proteinase to produce an intermediate peptide and then digesting the intermediate peptide with peptidase. The best results in WO 01/034828 are shown in example 2 where casein hydrolysed with papain and Lactobacillus helveticus derived peptidase gives a VPP yield is 25% and the IPP yield 97.5%, based on beta-casein. Assuming an average content of 30 wt. % beta-casein and 15 wt. % kappa-casein in skim milk powder, the yield of IPP yield of 97.5% based on beta-casein corresponds to a yield of 54% based on beta-casein and kappa casein together, as used herein. In the other examples lower yields are reported.